Simple STR profile interpretation

Question 1

Why do we conduct STR profile analysis

Answer 1

to provide a certain amount of confidence in your ability to determine whether an allele is actually an allele, a homozygote is actually a homozygote and a heterozygote is actually a heterozygote

Question 2

Why do we use statistics to apply analysis thresholds

Answer 2

to remove cognitive bias

Question 3

What can cause high risks in STR interpretation

Answer 3

1. complex results
2. approach is un-researched
3. operators are inexperienced
4. checking is conducted collaboratively

Question 4

What are the 5 main stages of simple profile interpretation

Answer 4

1. Check allele size standard
2. Check positive and negative controls
3. Check allele signal/ noise thresholds
4. check profile and allele balance
5. Check allele stutter

Question 5

What changes when more or less DNA is put into the reaction

Answer 5

the signal produced

Question 6

What are analytical thresholds set on

Answer 6

the average noise level across the baseline

Question 7

What is the limit of detection

Answer 7

average + 3STD

Question 8

What are the different channels of STR profiles created by

Answer 8

different wavelengths of light

Question 9

Name the 3 quality assurance practices employed during allele sizing to minimise inaccuracy

Answer 9

1. Size standards run in each well during size separation
2. Allelic ladders run alongside samples
3. DNA controls (positive and negative)

Question 10

Explain how the use of a size standard during allele sizing will reduce inaccuracy

Answer 10

Sizing can be based off of a standard curve created using the size standards data

Question 11

Explain how allelic ladders can reduce inaccuracy when sizing unknown alleles

Answer 11

They are used to set allele bin windows which will contain all common alleles at known sizes

Question 12

What kind of positive control do we use in allele sizing

Answer 12

007 positive control

Question 13

How do we distinguish between peaks and background noise

Answer 13

1. apply a threshold to the data; peaks will fall above and noise will fall below
2. peaks should conform to allele bin windows

Question 14

Should the negative control STR profile contain any peaks

Answer 14

No - otherwise would show instance of amplification

Question 15

Why are homozygote peaks typically bigger than heterozygote peaks

Answer 15

as two alleles are contributing to the same peak

Question 16

What does homozygote balance mean

Answer 16

the homozygote peaks heights are more than 85% similar to each other

Question 17

What can cause poor heterozygote peak balance

Answer 17

decay or inhibition

Question 18

Why do we have quality control peaks

Answer 18

to highlight whether degradation or inhibition has occurred

Question 19

Name the 4 effects that can complicate interpretation

Answer 19

1. Stutter
2. Mixtures
3. Null alleles
4. Drop-out/ drop-in

Question 20

How do we calculate heterozygote balance

Answer 20

(area of second allele/ area of first allele) x100

Question 21

How do we calculate thresholds for heterozygote balance and stutter

Answer 21

by running internal validation studies specific to laboritries

Question 22

What causes the formation of stutter peaks

Answer 22

strand slippage during PCR

Question 23

All human STRs used in forensic analysis are…

Answer 23

tetra-nucleotide

Question 24

How can stutter peaks cause problems

Answer 24

1. Can be confused for an allele peak
2. Can contribute to the overall fluorescence of an allele peak if the alleles are neighbouring - affecting heterozygote balance

Question 25

What is stutter expected to be

Answer 25

<15%